The present invention concerns a double confocal scanning microscope having an illuminating beam path of a light source and a detection beam path of a detector.
EP 0 491 289 A1 discloses a double confocal scanning microscope in which the light of a light source is divided into two partial beams, and each partial beam is focused with the aid of a respective microscope objective onto a common specimen point. The two microscope objectives are arranged on different sides of the specimen plane that is common to them. At the specimen point, this interferometric illumination causes formation of an interference pattern that, in the case of constructive interference, exhibits a principal maximum and several secondary maxima. The illumination pattern in the specimen region and in the common microscope objective focus is also referred to as a double confocal illumination point spread function (illumination PSF). If only double confocal illumination is implemented, the term “type A double confocal scanning microscope” is applied. Detected light then emerges from the specimen illuminated by the illumination PSF; the detected light can be, for example, fluorescent, reflected, or transmitted light. If the optical path length difference between the two beam path segments is less than the coherence length of the detected light, the detected light can in turn interfere at the detection pinhole. The interfering or non-interfering detected light imaged by the microscope objectives forms an illumination pattern that is also referred to as the detection point spread function (detection PSF). If only double confocal detection needs to be implemented, for example because the specimen is illuminated with the light from only one partial beam, the term “type B double confocal scanning microscope” is applied. In situations in which both double confocal illumination and double confocal detection are implemented, the term “type C double confocal scanning microscope” is applied.
A double confocal illumination PSF and/or detection PSF exhibits secondary maxima that generally are arranged along the optical axis. As a result of the interferometric illumination or detection, increased axial resolution as compared to conventional (scanning) microscopy can be achieved with a double confocal scanning microscope.
The image of a specimen acquired with a double confocal scanning microscope principally exhibits a contribution that results from the principal maximum of the illumination PSF and/or detection PSF. Additionally superimposed on the image, however, are components that result from the secondary maxima of the illumination PSF and/or detection PSF. Since these image components are disruptive, they are generally removed from the acquired image later using reconstruction methods. Inverse filtration methods, which are implemented in the form of program modules on a computer, are primarily used in this context. The reconstruction methods can only be successfully applied, however, if the intensity of the secondary maxima is much less than 50% of the intensity of the principal maximum of the illumination PSF or detection PSF. If this condition is not met, either the noise component of the reconstructed image is too high, or the contributions of the secondary maxima cannot be completely removed from the image, so that “ghosts” of the imaged specimen structure remain in the image. Unequivocal specimen analysis and image interpretation can thereby be rendered difficult or even impossible.
It is therefore an object of the present invention to eliminate the problems of the reconstruction methods at their cause.